The present invention relates to xylose isomerase genes of Thermus bacteria, especially Thermus aquaticus (hereinafter abbreviated as T. aquaticus), xylose isomerase produced by genetic engineering using the genes, a process for preparation of a xylose isomerase and a process for preparation of highly concentrated fructose using the xylose isomerase.
Xylose isomerases are enzymes that catalyze the interconversion between D-xylose and D-xylulose, and can also convert glucose into fructose which is important in industry.
T. aquaticus is a thermophilic bacterium, deposited as Accession Number ATCC 27634. T. aquaticus is also referred to as Thermus thermophilus. It has been reported that T. aquaticus produces a xylose isomerase (see, Journal of General Microbiology (1990), 136, 679-686), which was proven by the inventors to be a different enzyme from the enzyme produced by the present method in molecular weight, N-terminal amino acid sequence and others. T. aquaticus is a thermophilic bacterium and its xylose isomerase has a higher optimal temperature and thermal stability than Clostridium thermohydrosulfuricum xylose isomerase which also has an excellent thermostability compared to other isomerases which are presently utilized industrially. The enzyme is different, and any process for preparation of xylose isomerase in a large scale which is useful on an industrial scale is not yet known in the art.
Thus, once the xylose isomerase gene of T. aquaticus is obtained, it is possible to produce the T. aquaticus xylose isomerase in large scale by genetic engineering techniques. However, the isolation, identification and cloning of the T. aquaticus xylose isomerase gene has not been accomplished. Therefor, there is a need to provide the xylose isomerase gene of T. aquaticus.
The temperature at which a high fructose corn syrup (HFCS) is produced is conventionally selected at 60.degree. C. based on the thermostability of the currently used isomerase. The fructose concentration in the product is dependent on the chemical equilibrium and is 42-45% in the case of the immobilized enzyme procedure at 60.degree. C. But, in practical use, if the fructose concentration could be increased up to 55%, it may be used as a food sweetener for soft drinks without a further increase in fructose concentration. Then, if the production temperature could be raised to 85.degree. C., the chemical equilibrium is shifted in the direction of more fructose production and it is possible to increase the fructose concentration to 55%.